The blood group substances, which are mostly determined by conventional agglutination methods, belong to the classical blood cell antigens. On the other hand, antigens of the granulocytes, lymphocytes, monocytes and thrombocytes are largely determined at the present time by means of fluorescence-labeled antibodies. Not only are blood cells assigned unambiguously to the individual cell classes on the basis of their immunological properties, but their functional state is also characterized on the same basis. In medical diagnostics, therefore, immunocytometric methods have become widespread. At the present time, they serve to differentiate different forms of leukemia and lymphocytes and identify and evaluate defects in the immune system.
Monoclonal antibodies, labeled with different fluorochromes, are used to identify certain antigenic epitopes. Individual cells can be detected directly with a fluorescence microscope or a flow cytometer to indicate the presence of certain antigens. In indirect labeling, it is not the primary antibody, which recognizes the epitope in question, which has a fluorescent label, but a second antibody, which is directed against the protein of the primary antibody and bound by this. The immunotyping of blood cells with fluorescing antibodies is based at the present time particularly on flow cytometry, which permits thousands of individual cells to be analyzed in less than one minute and the fluorescence properties, as well as the characteristic light-scattering properties are determined for each cell. The fluorescence measurement serves to quantify the cell-bound antibodies. On the other hand, the light-scattering properties reflect morphological characteristics of the individual classes of cells. With flow cytometry therefore, statistically confirmed results can be achieved, the correctness of which is based on the partially automated, simultaneous evaluation of several cellular characteristics.
Since the efficient method of flow cytometry has been used in medical diagnosis, it has become increasingly clear that immunocytometric results depend appreciably on the functional state of the cells investigated. The expression of the antigens investigated depends namely to a large extent on the activation state of the cells and the effects of handling and keeping the blood sample or the cell preparation. For example, antigens are destroyed after a few hours by cellular proteases. The results are affected qualitatively and quantitatively by such pre-analytical effects. In the case of very rapidly reacting cells, such as the thrombocytes, dramatic changes in different surfaces/antigens are unavoidable if the cells are not stabilized immediately when the sample is taken. These interfering effects are particularly critical when the blood cells must be worked up or isolated before the measurement.
Therefore, for immunological phenotyping of blood cells and particularly for the quantitative analysis of antigen expression, it would be of appreciable benefit if the sample could be stabilized so that the pre-analytical changes in the cell antigens to be investigated are precluded. Such a procedure is indispensable for most of the questions relating to thrombocytes and necessary to quantify the antigen expression of granulocytes, monocytes, lymphocytes and erythrocytes. For the last-mentioned cells, this is true particularly when the samples cannot or should not be analyzed immediately after they are obtained.